* Each gene you analyze requires a set of three oligos: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.

-

* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.

+

* Use Roche's [http://www.roche-applied-science.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10001&tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1#tab-3 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.

* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

Line 31:

Line 31:

** Gene targets + GAPD reference = 5

** Gene targets + GAPD reference = 5

** Replicates per reaction = 3

** Replicates per reaction = 3

-

** '''Wells need = 3 * 5 * 3 = 45'''

+

** '''Wells needed = 3 * 5 * 3 = 45'''

Line 73:

Line 73:

| '''PLATE LAYOUT'''<br>

| '''PLATE LAYOUT'''<br>

Variation 1<br> [[Image:Haynes_UPL_fig1.png|300px|Figure 1]] <br>

Variation 1<br> [[Image:Haynes_UPL_fig1.png|300px|Figure 1]] <br>

-

Variation 2<br> [[Image:Haynes_UPL_fig2.png|300px|Figure 1]]

+

Variation 2<br> [[Image:Haynes_UPL_fig2.png|300px|Figure 1]]<br>

+

Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the plate as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.

Wet bench: Add the appropriate combinations of of the master mixes to the 96-well plate

---> Run the reaction in the Light Cycler!

1. Design your primers

Each gene you analyze requires a set of three oligos: forward primer, reverse primer, and a UPL probe.

Use Roche's Assay Design Center to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.

The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

2. Set up a reaction list and plan the plate layout

How many reactions should I plan to run? Each experimental cDNA sample is a Template. The gene being detected is often referred to as a Gene Target. You should also include a reference Gene Target such as GAPD (a housekeeping gene that is always active, not expected to change). Each unique template and target combination requires its own reaction. You will also need to set up a no template control to observe the amount of background noise from that reaction.

Hypothetical example:

cDNA samples + a no template control = 3

Gene targets + GAPD reference = 5

Replicates per reaction = 3

Wells needed = 3 * 5 * 3 = 45

REACTION LIST

Template cDNA

Gene Target

Rxn 1:

treated cells

MPK14

Rxn 2:

treated cells

CBX8

Rxn 3:

treated cells

TNF

Rxn 4:

treated cells

NPPA

Rxn 5:

treated cells

GAPD (reference gene)

Rxn 6:

untreated cells

MPK14

Rxn 7:

untreated cells

CBX8

Rxn 8:

untreated cells

TNF

Rxn 9:

untreated cells

NPPA

Rxn 10:

untreated cells

GAPD (reference gene)

Rxn 11:

no template

MPK14

Rxn 12:

no template

CBX8

Rxn 13:

no template

TNF

Rxn 14:

no template

NPPA

Rxn 15:

no template

GAPD (reference gene)

PLATE LAYOUT

Variation 1
Variation 2
Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the plate as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.

A single plate contains 96 wells. To insure accuracy, three technical replicates per reaction (Rxn) are required

If you need more than 96 wells, you must split the experiment over multiple plates.

It is absolutely critical that you keep a reaction list and plate layout in your notes. Your plate set-up will probably vary for each run.

3. Reaction set-up: PCR master mixes for each Gene Target

Label one 1.5 mL tube per gene target

Make enough PCR master mix for your plate...

MPK14 is in Reactions 1, 6, and 11 = 3

Replicates per reaction = 3

Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10

The same needs to be done for CBX8, TNF, NPPA, and GAPD in separate tubes.